Antisense composition and method for treating muscle atrophy

ABSTRACT

A method and compound for treating skeletal muscle mass deficiency in a human subject are disclosed. The composition is an oligomer of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, contains between 10-40 nucleotide bases, has a base sequence effective to hybridize to an expression-sensitive region of processed or preprocessed human myostatin RNA transcript, identified, in its processed form, by SEQ ID NO:6, and is capable of uptake by target muscle cells in the subject. In practicing the method, the compound is administered in an amount and at a dosage schedule to produce an overall reduction in the level of serum myostatin measured in the patient, and preferably to bring the myostatin level within the a range determined for normal, healthy individuals.

This application is a continuation of U.S. patent application Ser. No.11/433,724, filed May 11, 2006, now allowed, which is acontinuation-in-part application of International Patent Application No.PCT/US2006/004797, filed Feb. 9, 2006, which claims the benefit of U.S.Provisional Application No. 60/651,574, filed Feb. 9, 2005, each ofwhich is incorporated by reference in its entirety.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 120178_(—)437C2_SEQUENCE_LISTING.txt. The textfile is about 7 KB, was created on Jan. 3, 2011, and is being submittedelectronically via EFS-Web.

FIELD OF THE INVENTION

This invention relates to compounds and methods for treatingmuscle-wasting disease conditions, and additionally, for impactingmuscle tissue development in mammalian subjects.

REFERENCES

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(2000). “Anti-proliferative    effects of steric blocking phosphorodiamidate morpholino antisense    agents directed against c-myc.” Antisense Nucleic Acid Drug Dev    10(3): 163-76.-   Kirk, S., et al., “Myostatin regulation during skeletal muscle    regulation, J. Cell Physiology, 2000, 184(3): 356-363.-   Lappalainen, K., A. Urtti, et al. (1994). “Cationic liposomes    improve stability and intracellular delivery of antisense    oligonucleotides into CaSki cells.” Biochim Biophys Acta 1196(2):    201-8.-   Lesnikowski, Z. J., M. Jaworska, et al. (1990). “Octa(thymidine    methanephosphonates) of partially defined stereochemistry: synthesis    and effect of chirality at phosphorus on binding to    pentadecadeoxyriboadenylic acid.” Nucleic Acids Res 18(8): 2109-15.-   Lou, X., K. L. Garrett, et al. (2001). “Synthetic hydrogels as    carriers in antisense therapy: preliminary evaluation of an    oligodeoxynucleotide covalent conjugate with a copolymer of    1-vinyl-2-pyrrolidinone and 2-hydroxy-ethyl methacrylate.” J    Biomater Appl 15(4): 307-20.-   McPherron, A. C., A. M. Lawler, et al. (1997). “Regulation of    skeletal muscle mass in mice by a new TGF-beta superfamily member.”    Nature 387(6628): 83-90.-   McPherron, A. C. and S.-J. Lee (1997). “Double muscling in cattle    due to mutations in the myostatin gene.” PNAS 94(23): 12457-12461.-   Mertes, M. P. and E. A. Coats (1969). “Synthesis of carbonate    analogs of dinucleosides. 3′-Thymidinyl 5′-thymidinyl carbonate,    3′-thymidinyl 5′-(5-fluoro-2′-deoxyuridinyl) carbonate, and    3′-(5-fluoro-2′-deoxyuridinyl) 5′-thymidinyl carbonate.” J Med Chem    12(1): 154-7.-   Schulte, J. N., et al. “Effects of resistance training on the rate    of muscle protein synthesis in frail elderly people, Int. J. Sport    Nutrition and Exercise Metab, 2001, 11Supp, pp 111-118.-   Stein, D., E., et al. (1997). “A specificity comparison of four    antisense types: morpholino, 2′-O-methyl RNA, DNA, and    phosphorothioate DNA.” Antisense Nucleic Acid Drug Dev 7(3): 151-7.-   Toulme, J. J., R. L. Tinevez, et al. (1996). “Targeting RNA    structures by antisense oligonucleotides.” Biochimie 78(7): 663-73.-   Wallace, J. I. and R. S. Schwartz (2002). “Epidemiology of weight    loss in humans with special reference to wasting in the elderly.”    Int J Cardiol 85(1): 15-21.-   Williams, A. S., J. P. Camilleri, et al. (1996). “A single    intra-articular injection of liposomally conjugated methotrexate    suppresses joint inflammation in rat antigen-induced arthritis.” Br    J Rheumatol 35(8): 719-24.-   Yarasheski, K. E., et al., Serum myostatin-immunoreactive protein is    increased in 60-92 year old women and men with muscle wasting,” J.    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BACKGROUND OF THE INVENTION

Myostatin, or growth/differentiation factor 8 (GDF-8), belongs to thetransforming growth factor-β (TGF-β) superfamily (McPherron, Lawler etal. 1997). The human myostatin gene has been cloned (Gonzalez-Cadavid,Taylor et al. 1998), and it has been reported that myostatin is largelyexpressed in human skeletal muscle and plays an essential role innegatively regulating the growth and development of skeletal muscle(Gonzalez-Cadavid, Taylor et al. 1998).

Knock-out mice provided the first evidence that myostatin plays a keyrole in negatively regulating muscle development (McPherron, Lawler etal. 1997). The myostatin null mice were normal except that they weresignificantly larger than wild-type mice and had a large and widespreadincrease in skeletal muscle mass. Furthermore, it was also determinedthat two breeds of cattle, with the heritable characteristic ofincreased muscle mass, have mutations in the myostatin coding sequence(McPherron and Lee 1997). Furthermore, the serum and intramuscularconcentrations of myostatin are increased in HIV-infected men withmuscle wasting compared with healthy men (Gonzalez-Cadavid, Taylor etal. 1998). These data support the role of myostatin as a negativeregulator of skeletal muscle growth in adult men and as a contributor tomuscle wasting in HIV-infected men.

Heretofore, methods for treating muscle-wasting conditions and/orenhancing muscle mass in mammals by manipulating myostatin levels havebeen proposed. For example, U.S. Pat. Nos. 6,103,466 and 6,617,440, andU.S. published patent application 20030074680 A1 disclose methods forinhibiting levels of expressed myostatin by administering to a human oranimal subject, an antisense compound against the myostatin transcript.To date, there is no evidence that such approaches have succeeded orwould succeed as disclosed, or how one would select and monitor subjectsfor and during treatment. There is thus a need for a treatment methodfor effectively treating muscle wasting as a result of a condition suchas paralysis or disease state such as, for example, aging, acquiredimmune deficiency syndrome, multiple sclerosis, and cancer. There isalso a need for an antisense agent that can effectively accumulate intarget muscle cells, e.g., with oral administration, and inhibitmyostatin expression in muscle cells.

Methods for enhancing muscle mass in meat-bearing animals, byadministering anti-myostatin antisense agents, have also been proposed.However, to date such approaches have not proven practical because ofpoor uptake of the agents and/or inability to administer the agentsorally. It would thus be desirable to provide an agent that could besupplied orally, e.g., in animal feed, to enhance muscle mass inmeat-bearing animals.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, an antisense composition for usein increasing skeletal muscle mass in a human subject. The compositionincludes a substantially uncharged antisense compound (i) composed ofmorpholino subunits and phosphorus-containing intersubunit linkagesjoining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon ofan adjacent subunit; (ii) capable of uptake by target muscle cells inthe subject; (iii) containing between 10-40 nucleotide bases; and (iv)having a base sequence effective to hybridize to an expression-sensitiveregion of processed or preprocessed human myostatin RNA transcript,identified, in its processed form, by SEQ ID NO:6.

The morpholino subunits in the compound may be joined byphosphorodiamidate linkages, in accordance with the structure:

where Y₁═O, Z═O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkylamino, including dialkylamino, e.g., where X═NR₂, each R isindependently hydrogen or methyl. The compound may be composed ofmorpholino subunits linked with the uncharged linkages described aboveinterspersed with linkages that are positively charged at physiologicalpH. The total number of positively charged linkages is between 2 and nomore than half of the total number of linkages. The positively chargedlinkages have the structure above, where X is 1-piperazine.

The antisense compound in the composition may be conjugated to anarginine-rich polypeptide effective to promote uptake of the compoundinto target muscle cells. An exemplary arginine rich peptide has one ofthe sequences identified as SEQ ID NOS:7-9. The arginine-rich peptidemay be covalently coupled at its C terminus to the 5′ end of theantisense compound. Where the antisense compound is effective tohybridize to a target region at or adjacent the start site of theprocessed human myostatin transcript, the compound has a base sequencethat is complementary to a target region containing at least 12contiguous bases in a processed human myostatin transcript identified bySEQ ID NO:10, and formation of the heteroduplex in step (c) is effectiveto block translation of said processed transcript. An exemplaryantisense sequence includes the base sequence identified by the sequenceSEQ ID NO:1.

Where the antisense compound is effective to hybridize to a splice siteof preprocessed human myostatin transcript, it has a base sequence thatis complementary to at least 12 contiguous bases of a splice site in apreprocessed human myostatin transcript, and formation of theheteroduplex in step (c) is effective to block processing of apreprocessed myostatin transcript to produce a full-length, processedmyostatin transcript. The splice site in the preprocessed myostatintranscript may have one of the sequences identified as SEQ ID NOS:11-14. Exemplary antisense sequences are those identified by SEQ ID NOS:2-5.

In another aspect, the invention includes a method for treating loss ofskeletal muscle mass in a human subject. The steps in the method entail(a) measuring blood or tissue levels of myostatin in the subject, (b)administering to the subject, a myostatin-expression-inhibiting amountof the antisense composition described above, (c) by this administering,forming within target muscle cells in the subject, a base-pairedheteroduplex structure composed of human myostatin RNA transcript andthe antisense compound and having a Tm of dissociation of at least 45°C., thereby inhibiting expression of myostatin in said cells, (d) at aselected time following administering the antisense compound, measuringa blood or tissue level of myostatin in the subject, and (e) repeatingthe administering, using the myostatin levels measured in (d) to adjustthe dose or dosing schedule of the amount of antisense compoundadministered, if necessary, so as to reduce measured levels of myostatinover those initially measured and maintain such levels of myostatinmeasured in step (d) within a range determined for normal, healthyindividuals.

In one general embodiment, the myostatin value measured in step (a) isabove a selected threshold for normal healthy people. The administeringand measuring steps are preferably carried out over a selected period ofat least 2 weeks. The administering may be by oral route.

Also forming part of the invention is a method of measuring myostatinexpression levels in a mammalian subject, by (a) administering to thesubject, a myostatin-expression-inhibiting amount of the substantiallyuncharged antisense compound of the type described above, (b) withinabout 8-72 hours following the administering, analyzing a body-fluidsample obtained from the subject for the presence of a heteroduplexcomposed of the antisense compound and a complementary region of saidmyostatin RNA transcript, to determine the concentration of transcriptin said sample.

Also disclosed is a feed composition for a meat-producing animal,composed of a feed substance, and mixed therewith, a substantiallyuncharged antisense compound of the type described above.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures and examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows several preferred morpholino-type subunits having 5-atom(A), six-atom (B) and seven-atom (C-D) linking groups suitable forforming polymers.

FIGS. 2A-D show the repeating subunit segment of exemplary uncharged,morpholino oligonucleotides having phosphorus-containing linkages,designated A through D, constructed using subunits A-D, respectively, ofFIG. 1. FIG. 2E is another example of an uncharged linkage type in anoligonucleotide analog. FIG. 2F is an example of a preferred charged,cationic linkage.

FIG. 3 shows the synthetic steps to produce subunits used to produce+PMO containing the (1-piperazino) phosphinylideneoxy cationic linkageas shown in FIG. 2F.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise.

The terms “antisense oligonucleotides,” “antisense oligomer,” and“antisense compound” are used interchangeably and refer to a compoundhaving a sequence of nucleotide bases and a subunit-to-subunit backbonethat allows the antisense oligomer to hybridize to a target sequence inRNA by Watson-Crick base pairing, to form an RNA:oligomer heteroduplexwithin the target sequence. The antisense oligonucleotide includes asequence of purine and pyrimidine heterocyclic bases, supported by abackbone, which are effective to hydrogen-bond to corresponding,contiguous bases in a target nucleic acid sequence. The backbone iscomposed of subunit backbone moieties supporting the purine andpyrimidine heterocyclic bases at positions that allow such hydrogenbonding. These backbone moieties are cyclic moieties of 5 to 7 atoms inlength, linked together by phosphorous-containing linkages one to threeatoms long.

A substantially uncharged, phosphorus containing backbone in anoligonucleotide analog is one in which a majority of the subunitlinkages, e.g., between 50-100%, are uncharged at physiological pH, andcontain a single phosphorous atom. The analog contains between 12 and 40subunits, typically about 15-25 subunits, and preferably about 18 to 25subunits. The analog may have exact sequence complementarity to thetarget sequence or near complementarity, as defined below.

A “subunit” of an oligonucleotide analog refers to one nucleotide (ornucleotide analog) unit of the analog. The term may refer to thenucleotide unit with or without the attached intersubunit linkage,although, when referring to a “charged subunit”, the charge typicallyresides within the intersubunit linkage (e.g. a phosphate orphosphorothioate linkage). A “morpholino” oligonucleotide refers to apolymeric molecule having a backbone which supports bases capable ofhydrogen bonding to typical polynucleotides, wherein the polymer lacks apentose sugar backbone moiety, and more specifically a ribose backbonelinked by phosphodiester bonds which is typical of nucleotides andnucleosides, but instead contains a ring nitrogen with coupling throughthe ring nitrogen. A preferred “morpholino” oligonucleotide is composedof morpholino subunit structures of the form shown in FIG. 1A-1D, where(i) the structures are linked together by phosphorous-containinglinkages, one to three atoms long, joining the morpholino nitrogen ofone subunit to the 5′ exocyclic carbon of an adjacent subunit, and (ii)Pi and Pj are purine or pyrimidine base-pairing moieties effective tobind, by base-specific hydrogen bonding, to a base in a polynucleotide.Exemplary structures for antisense oligonucleotides for use in theinvention include the morpholino subunit types shown in FIGS. 1A-1D,with the uncharged, phosphorous-containing linkages shown in FIGS.2A-2D. The purine or pyrimidine base-pairing moiety is typicallyadenine, cytosine, guanine, uracil, thymine or inosine. The synthesis,structures, and binding characteristics of morpholino oligomers aredetailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506,5,166,315, 5,521,063, and 5,506,337, all of which are incorporatedherein by reference.

A preferred morpholino oligomer is a phosphorodiamidate-linkedmorpholino oligomer, referred to herein as a PMO. Such oligomers arecomposed of morpholino subunit structures such as shown in FIG. 2B,where X═NH₂, NHR, or NR₂ (where R is lower alkyl, preferably methyl),Y═O, and Z═O, and Pi and Pj are purine or pyrimidine base-pairingmoieties effective to bind, by base-specific hydrogen bonding, to a basein a polynucleotide, as seen in FIG. 2E. Also preferred are morpholinooligomers where the phosphordiamidate linkages are uncharged linkages asshown in FIG. 2E interspersed with cationic linkages as shown in FIG. 2Fwhere, in FIG. 2B, X=1-piperazino. In another FIG. 2B embodiment,X=lower alkoxy, such as methoxy or ethoxy, Y═NH or NR, where R is loweralkyl, and Z═O.

As used herein, an oligonucleotide or antisense oligomer “specificallyhybridizes” to a target polynucleotide if the oligomer hybridizes to thetarget under physiological conditions, with a thermal melting point (Tm)substantially greater than 37° C., preferably at least 45° C., andtypically 50° C.-80° C. or higher. Such hybridization preferablycorresponds to stringent hybridization conditions, selected to be about10° C., and preferably about 50° C. lower than the Tm for the specificsequence at a defined ionic strength and pH. At a given ionic strengthand pH, the Tm is the temperature at which 50% of a target sequencehybridizes to a complementary polynucleotide.

Polynucleotides are described as “complementary” to one another whenhybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides. A double-stranded polynucleotide can be“complementary” to another polynucleotide, if hybridization can occurbetween one of the strands of the first polynucleotide and the second.Complementarity (the degree that one polynucleotide is complementarywith another) is quantifiable in terms of the proportion of bases inopposing strands that are expected to form hydrogen bonds with eachother, according to generally accepted base-pairing rules. An antisensecompound may be complementary to a target region of a target transcripteven if the two bases sequences are not 100% complementary, as long asthe heteroduplex structure formed between the compound and transcripthas the desired Tm stability.

As used herein the term “analog” with reference to an oligomer means asubstance possessing both structural and chemical properties similar tothose of the reference oligomer.

As used herein, a first sequence is an “antisense sequence” or“targeting sequence” with respect to a second sequence or “targetsequence” if a polynucleotide whose sequence is the first sequencespecifically binds to, or specifically hybridizes with, the secondpolynucleotide sequence under physiological conditions.

As used herein, “effective amount” relative to an antisense oligomerrefers to the amount of antisense oligomer administered to a subject,either as a single dose or as part of a series of doses that areeffective to inhibit expression of a selected target nucleic acidsequence.

As used herein, an “expression-sensitive region” of a processed orpreprocessed mRNA transcript refers to either (i) a region including oradjacent the AUG start site of a processed transcript, where formationof an antisense-transcript heteroduplex is effective to inhibittranslation of the transcript or (ii) a region including or adjacent adonor or acceptor splice site junction in a preprocessed transcript,where formation of an antisense-transcript heteroduplex is effective toinhibit formation of a full-length processed transcript, either becauseone or more exons that would normally be included in the transcript havebeen deleted or because the transcript has been truncated at the targetsplice site.

An agent is “actively taken up by mammalian cells” when the agent canenter the cell by a mechanism other than passive diffusion across thecell membrane. The agent may be transported, for example, by “activetransport”, referring to transport of agents across a mammalian cellmembrane by e.g. an ATP-dependent transport mechanism, or by“facilitated transport”, referring to transport of antisense agentsacross the cell membrane by a transport mechanism that requires bindingof the agent to a transport protein, which then facilitates passage ofthe bound agent across the membrane. For both active and facilitatedtransport, the oligonucleotide compound has a substantially unchargedbackbone, as defined below. In addition, the analog may be conjugated,e.g., at its 5′ or 3′ end, to an arginine rich peptide, e.g., the HIVTAT protein, or polyarginine, to facilitate transport into the targethost cell, as discussed below.

As used herein, the term “myostatin antisense oligomer” refers to anuclease-resistant phosphorus-linked morpholino antisense oligomerhaving high affinity for (i.e., which “specifically hybridizes to”) acomplementary or near-complementary human myostatin nucleic acidsequence.

As used herein “treatment” of an individual or a cell is any type ofintervention used in an attempt to alter the natural course of theindividual or cell. Treatment includes, but is not limited to,administration of e.g., a pharmaceutical composition, and may beperformed either prophylactically, or subsequent to the initiation of apathologic event or contact with an etiologic agent.

II. Antisense Composition for use in Practicing the Invention

Antisense compound. Antisense compounds in accordance with the presentinvention are substantially uncharged antisense compounds (i) composedof morpholino subunits and phosphorus-containing intersubunit linkagesjoining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon ofan adjacent subunit; (ii) capable of uptake by target muscle cells inthe subject; (iii) containing between 10-40 nucleotide bases; (iv)having a base sequence effective to hybridize to an expression-sensitiveregion of processed or preprocessed human myostatin RNA transcript,identified, in its processed form, by SEQ ID NO:6; to form aheteroduplex complex having a Tm substantially greater than 37° C.,preferably at least 45° C., and (vi) nuclease resistance.

In addition, the antisense compound may have the capability for activeor facilitated transport as evidenced by (i) competitive binding with aphosphorothioate antisense oligomer, and/or (ii) the ability totransport a detectable reporter into target cells.

Candidate antisense oligomers may be evaluated, according to well knownmethods, for acute and chronic cellular toxicity, such as the effect onprotein and DNA synthesis as measured via incorporation of 3H-leucineand 3H-thymidine, respectively. In addition, various controloligonucleotides, e.g., control oligonucleotides such as sense, nonsenseor scrambled antisense sequences, or sequences containing mismatchedbases, in order to confirm the specificity of binding of candidateantisense oligomers. The outcome of such tests is important indiscerning specific effects of antisense inhibition of gene expressionfrom indiscriminate suppression. Accordingly, sequences may be modifiedas needed to limit non-specific binding of antisense oligomers tonon-target nucleic acid sequences.

Heteroduplex formation. The effectiveness of a given antisense compoundin forming a heteroduplex with the target mRNA may be determined byscreening methods known in the art. For example, the oligomer isincubated in a cell culture containing an mRNA preferentially expressedin activated lymphocytes, and the effect on the target mRNA is evaluatedby monitoring the presence or absence of (1) heteroduplex formation withthe target sequence and non-target sequences using procedures known tothose of skill in the art, (2) the amount of the target mRNA expressedby activated lymphocytes, as determined by standard techniques such asRT-PCR or Northern blot, (3) the amount of protein transcribed from thetarget mRNA, as determined by standard techniques such as ELISA orWestern blotting. (See, for example, (Pari, Field et al. 1995; Anderson,Fox et al. 1996).

Uptake into cells. A second test measures cell transport, by examiningthe ability of the test compound to transport a labeled reporter, e.g.,a fluorescence reporter, into cells, e.g., cultured myocytes. The cellsare incubated in the presence of labeled test compound, added at a finalconcentration between about 10-300 nM. After incubation for 30-120minutes, the cells are examined, e.g., by microscopy or FACS analysis,for intracellular label. The presence of significant intracellular labelis evidence that the test compound is transported by facilitated oractive transport.

In one embodiment of the invention, uptake into cells is enhanced byadministering the antisense compound in combination with anarginine-rich peptide linked to the 5′ or 3′ end of the antisenseoligomer. The peptide is typically 8-16 amino acids and consists of amixture of arginine, and other amino acids including phenylalanine andcysteine, as discussed further below.

RNAse resistance. Two general mechanisms have been proposed to accountfor inhibition of expression by antisense oligonucleotides (Agrawal,Mayrand et al. 1990; Bonham, Brown et al. 1995; Boudvillain, Guerin etal. 1997). In the first, a heteroduplex formed between theoligonucleotide and the viral RNA acts as a substrate for RNaseH,leading to cleavage of the RNA. Oligonucleotides belonging, or proposedto belong, to this class include phosphorothioates, phosphotriesters,and phosphodiesters (unmodified “natural” oligonucleotides). Suchcompounds expose the RNA in an oligomer:RNA duplex structure tohydrolysis by RNaseH, and therefore loss of function.

A second class of oligonucleotide analogs, termed “steric blockers” or,alternatively, “RNaseH inactive” or “RNaseH resistant”, have not beenobserved to act as a substrate for RNaseH, and act by stericallyblocking target RNA nucleocytoplasmic transport, splicing, translation,or replication. This class includes methylphosphonates (Toulme, Tinevezet al. 1996), morpholino oligonucleotides, peptide nucleic acids(PNA's), certain 2′-O-allyl or 2′-β-alkyl modified oligonucleotides(Bonham, Brown et al. 1995), and N3′→P5′ phosphoramidates (Ding,Grayaznov et al. 1996; Gee, Robbins et al. 1998).

A test oligomer can be assayed for its RNaseH resistance by forming anRNA:oligomer duplex with the test compound, then incubating the duplexwith RNaseH under a standard assay conditions, as described (Stein,Foster et al. 1997). After exposure to RNaseH, the presence or absenceof intact duplex can be monitored by gel electrophoresis or massspectrometry.

In vivo uptake. In accordance with another aspect of the invention,there is provided a simple, rapid test for confirming that a givenantisense oligomer type provides the required characteristics notedabove, namely, high Tm, ability to be actively taken up by the hostcells, and substantial resistance to RNaseH. This method is based on thediscovery that a properly designed antisense compound will form a stableheteroduplex with the complementary portion of the RNA target whenadministered to a mammalian subject, and the heteroduplex subsequentlyappears in the urine (or other body fluid). Details of this method arealso given in co-owned U.S. Pat. No. 6,365,351 for “Non-Invasive Methodfor Detecting Target RNA,” the disclosure of which is incorporatedherein by reference.

Briefly, a test morpholino oligomer having an unchargedphosphorus-containing backbone to be evaluated, and having a basesequence targeted against a known myostatin RNA sequence (notnecessarily an expression-sensitive region of the RNA transcript), isinjected into a mammalian subject. Several hours (typically 8-72) afteradministration, the urine is assayed for the presence of theantisense-RNA heteroduplex. If heteroduplex is detected, the backbone issuitable for use in the antisense oligomers of the present invention.

The test oligomer may be labeled, e.g. by a fluorescent or a radioactivetag, to facilitate subsequent analyses, if it is appropriate for themammalian subject. The assay can be in any suitable solid-phase or fluidformat. Generally, a solid-phase assay involves first binding theheteroduplex analyte to a solid-phase support, e.g., particles or apolymer or test-strip substrate, and detecting the presence/amount ofheteroduplex bound. In a fluid-phase assay, the analyte sample istypically pretreated to remove interfering sample components. If theoligomer is labeled, the presence of the heteroduplex is confirmed bydetecting the label tags. For non-labeled compounds, the heteroduplexmay be detected by immunoassay if in solid phase format or by massspectroscopy or other known methods if in solution or suspension format.

Structural features. As detailed above, the antisense oligomer has abase sequence directed to a targeted portion of a cellular gene,preferably the region at or adjacent the start codon or a processedtranscript or a region at or adjacent a splice site junction of themyostatin mRNA or preprocessed transcript. In addition, the oligomer isable to effectively inhibit expression of the targeted gene whenadministered to a host cell, e.g. in a mammalian subject. Thisrequirement is met when the oligomer compound (a) has the ability to betaken up by muscle cells, and (b) once taken up, form a duplex with thetarget RNA with a Tm greater than about 45° C., preferably greater than50° C.

The ability to be taken up selectively by activated immune cellsrequires, in part, that the oligomer backbone be substantiallyuncharged. The ability of the oligomer to form a stable duplex with thetarget RNA will depend on the oligomer backbone, the length and degreeof complementarity of the antisense oligomer with respect to the target,the ratio of G:C to A:T base matches, and the positions of anymismatched bases. The ability of the antisense oligomer to resistcellular nucleases promotes survival and ultimate delivery of the agentto the cell cytoplasm.

Antisense oligonucleotides of 15-20 bases are generally long enough tohave one complementary sequence in the mammalian genome. In addition,antisense compounds having a length of at least 12, typically at least15 nucleotides in length hybridize well with their target mRNA. Due totheir hydrophobicity, antisense oligonucleotides tend to interact wellwith phospholipid membranes, and it has been suggested that followingthe interaction with the cellular plasma membrane, oligonucleotides areactively transported into living cells (Loke, Stein et al. 1989;Yakubov, Deeva et al. 1989; Anderson, Xiong et al. 1999).

Morpholino oligonucleotides, particularly phosphoramidate- orphosphorodiamidate-linked morpholino oligonucleotides have been shown tohave high binding affinities for complementary or near-complementarynucleic acids. Morpholino oligomers also exhibit little or nonon-specific antisense activity, afford good water solubility, areimmune to nucleases, and are designed to have low production costs(Summerton and Weller 1997).

Morpholino oligonucleotides (including antisense oligomers) aredetailed, for example, in co-owned U.S. Pat. Nos. 5,698,685, 5,217,866,5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, and 5,506,337,all of which are expressly incorporated by reference herein

As noted above, the antisense oligomers for use in practicing theinvention are composed of morpholino subunits of the form shown in theabove cited patents, where (i) the morpholino groups are linked togetherby uncharged phosphorus-containing linkages, one to three atoms long,joining the morpholino nitrogen of one subunit to the 5′ exocycliccarbon of an adjacent subunit, and (ii) the base attached to themorpholino group is a purine or pyrimidine base-pairing moiety effectiveto bind, by base-specific hydrogen bonding, to a base in apolynucleotide. The purine or pyrimidine base-pairing moiety istypically adenine, cytosine, guanine, uracil or thymine. Preparation ofsuch oligomers is described in detail in U.S. Pat. No. 5,185,444(Summerton et al., 1993), which is hereby incorporated by reference inits entirety. As shown in this reference, several types of nonioniclinkages may be used to construct a morpholino backbone.

Exemplary subunit structures for antisense oligonucleotides of theinvention include the morpholino subunit types shown in FIGS. 1A-D, eachlinked by an uncharged, phosphorous-containing subunit linkage, as shownin FIGS. 2A-2D, respectively. In these figures, the X moiety pendantfrom the phosphorous may be any of the following: fluorine; an alkyl orsubstituted alkyl; an alkoxy or substituted alkoxy; a thioalkoxy orsubstituted thioalkoxy; or, an unsubstituted, monosubstituted, ordisubstituted nitrogen, including cyclic structures. Alkyl, alkoxy andthioalkoxy preferably include 1-6 carbon atoms, and more preferably 1-4carbon atoms. Monosubstituted or disubstituted nitrogen preferablyrefers to lower alkyl substitution, and the cyclic structures arepreferably 5- to 7-membered nitrogen heterocycles optionally containing1-2 additional heteroatoms selected from oxygen, nitrogen, and sulfur. Zis sulfur or oxygen, and is preferably oxygen.

FIG. 1A shows a phosphorous-containing linkage which forms the five atomrepeating-unit backbone shown in FIG. 2A, where the morpholino rings arelinked by a 1-atom phosphoamide linkage. Subunit B in FIG. 1B isdesigned for 6-atom repeating-unit backbones, as shown in FIG. 2B. InFIG. 1B, the atom Y linking the 5′ morpholino carbon to the phosphorousgroup may be sulfur, nitrogen, carbon or, preferably, oxygen. The Xmoiety pendant from the phosphorous may be any of the following:fluorine; an alkyl or substituted alkyl; an alkoxy or substitutedalkoxy; a thioalkoxy or substituted thioalkoxy; or, an unsubstituted,monosubstituted, or disubstituted nitrogen, including cyclic structures.Z is sulfur or oxygen, and is preferably oxygen. Particularly preferredmorpholino oligonucleotides include those composed of morpholino subunitstructures of the form shown in FIG. 2B, where X is an amine or alkylamine of the form X═NR₂, where R is independently H or CH₃, that iswhere X═NH₂, X═NHCH₃ or X═N(CH₃)₂, Y═O, and Z═O.

Subunits C-D in FIGS. 1C-D are designed for 7-atom unit-length backbonesas shown for structures in FIGS. 2C and D. In Structure C, the X moietyis as in Structure B, and the moiety Y may be methylene, sulfur, orpreferably oxygen. In Structure D, the X and Y moieties are as inStructure B. In all subunits depicted in FIGS. 1 and 2, each Pi and Pjis a purine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide, and ispreferably selected from adenine, cytosine, guanine and uracil.

As noted above, the substantially uncharged oligomer may advantageouslyinclude a limited number of charged linkages, e.g. up to about 1 perevery 5 uncharged linkages. In the case of the morpholino oligomers,such a charged linkage may be a linkage as represented by any of FIGS.2A-D, preferably FIG. 2B, where X is oxide (—O—) or sulfide (—S—).

Also shown is a cationic linkage in FIG. 2F wherein the nitrogen pendantto the phosphate atom in the linkage of FIG. 2E is replaced with a1-piperazino structure. The method for synthesizing the 1-piperazinogroup linkages is described below with respect to FIG. 3.

Preferred Antisense Targets. In the method and composition of theinvention, the antisense oligomer is designed to hybridize to anexpression-sensitive region of the myostatin nucleic acid sequence,under physiological conditions with a Tm substantially greater than 37°C., e.g., at least 50° C. and preferably 60° C. to 80° C. The antisensecompound is designed to have high-binding affinity to the nucleic acidand may be 100% complementary to the myostatin target sequence or mayinclude mismatches, e.g., to accommodate allelic variants, as long asthe heteroduplex formed between the oligomer and myostatin targetsequence is sufficiently stable to withstand the action of cellularnucleases and other modes of degradation during its transit from cell tobody fluid. Mismatches, if present, are less destabilizing toward theend regions of the hybrid duplex than in the middle. The number ofmismatches allowed will depend on the length of the oligomer, thepercentage of G:C base pair in the duplex and the position of themismatch(es) in the duplex, according to well understood principles ofduplex stability.

Although such an antisense oligomer is not necessarily 100%complementary to the myostatin target sequence, it is effective tostably and specifically bind to the target sequence such that expressionof myostatin is modulated. The appropriate length of the oligomer toallow stable, effective binding combined with good specificity is about840 nucleotide base units, and preferably about 12-25 nucleotides.Oligomer bases that allow degenerate base pairing with target bases arealso contemplated, assuming base-pair specificity with the target ismaintained.

In one preferred approach, the target for modulation of gene expressionusing the antisense methods of the present invention comprises asequence spanning or adjacent to the mRNA translational start codon formyostatin. In an alternative preferred approach, a splice acceptor ordonor region of preprocessed myostatin mRNA is targeted. It will beunderstood that other regions of myostatin mRNA may be targeted,including one or more of, an initiator or promoter site, an intron orexon junction site, a 3′-untranslated region, and a 5′-untranslatedregion. It will be further understood that both spliced and unsplicedRNA may serve as the template for design of antisense oligomers for usein the methods of the invention (See, e.g., Hudziak, Summerton et al.2000).

Table 1 below lists exemplary target regions in the human myostatin gene(SEQ ID NOS:10-14). The translational start site target (MSTN-AUG)covers a region from −28 to +24 relative to the A residue of the ATGstart codon (shown in bold). The Nucleotide Region (Nct. Region) isrelative to a human bacterial artificial chromosome sequence (GenBankAccession No. AC073120) that contains the myostatin gene. For the splicedonor (SD) and splice acceptor (SA) targets the splice site is indicatedwithin the sequence with “I”. The specific target regions for theantisense oligomers listed in Table 2 are contained within the targetsequences shown in Table 1 and are exemplary. It is fully anticipatedthat alternative antisense oligomers that target different sequenceswithin those shown in Table 1 would function to effectively decrease theexpression of the myostatin gene.

TABLE 1 Human Myostatin Target Regions Nct.  SEQ  Target Sequence RegionID Name (5′ to 3′) (AC073120) NO MSTN-AUG GAAAAAAGATTATATTGATTTTAAAA29692-29743 10 TCATGCAAAAACTGCAACTCTGTGTT MSTN-SD1ACAATCATTACCATGCCTACAGAGT/ 29318-29367 11 GTAAGTAGTCCTATTAGTGTATATCMSTN-SA2 CTTTTCTTTTCTTATTCATTTATAG/ 27530-27579 12CTGATTTTCTAATGCAAGTGGATGG MSTN-SD2 CCCAGGACCAGGAGAAGATGGGCTG/27156-27205 13 GTAAGTGATAACTGAAAATAACATT MSTN-SA3TGATTGTTCTTTCCTTTTCAAACAG/ 24733-24782 14 AATCCGTTTTTAGAGGTCAAGGTAA

Exemplary antisense oligomers to myostatin and their targets areprovided in Table 2, below. The complement to the myostatintranslational start codon is indicated in bold.

TABLE 2 Exemplary Myostatin Antisense Oligomers Antisense Oligomer SEQ(5′ to 3′) Target GenBank ID Name Targeting Sequence Ncts. Acc. # NO.MSTN-AUG GAGTTGCAGTTTTTGCATG 133-151 AF104922 1 MSTN-SD1ACTCTGTAGGCATGGTAATG 487-506 AF104922 2 MSTN-SD2 CAGCCCATCTTCTCCTGG730-747 AF104922 3 MSTN-SA2 CACTTGCATTAGAAAATCAG 507-526 AF104922 4MSTN-SA3 CTTGACCTCTAAAAACGGATT 881-901 AF104922 5

In exemplary embodiments of the invention, the antisense oligomer is aPMO containing the sequences presented as SEQ ID NOS:1-5.

Oligomers as long as 40 bases may be suitable, where at least a minimumnumber of bases, e.g., 12 bases, are complementary to the targetsequence. In general, however, facilitated or active uptake in cells isoptimized at oligomer lengths less than about 30, preferably less than25. For PMO oligomers, described further below, an optimum balance ofbinding stability and uptake generally occurs at lengths of 15-22 bases.The effectiveness of a given antisense oligomer molecule in forming aheteroduplex with the target RNA may be determined by screening methodsknown in the art. For example, the oligomer is incubated a cell cultureexpressing myostatin and the effect on the target RNA is evaluated bymonitoring the presence or absence of (1) heteroduplex formation withthe target sequence and non-target sequences using procedures known tothose of skill in the art, (2) the amount of myostatin mRNA, asdetermined by standard techniques such as RT-PCR or Northern blot, or(3) the amount of myostatin protein, as determined by standardtechniques such as ELISA or immunoblot (e.g. Western blot).

The antisense activity of the oligomer may be enhanced by using amixture of uncharged and cationic phosphorodiamidate linkages as shownin FIGS. 2G and 2F. The total number of cationic linkages in theoligomer can vary from 1 to 10, and be interspersed throughout theoligomer. Preferably the number of charged linkages is at least 2 and nomore than half the total backbone linkages, e.g., between 2-8 positivelycharged linkages, and preferably each charged linkages is separatedalong the backbone by at least one, preferably at least two unchargedlinkages. The antisense activity of various oligomers can be measured invitro by fusing the oligomer target region to the 5′ end a reporter gene(e.g. firefly luciferase) and then measuring the inhibition oftranslation of the fusion gene mRNA transcripts in cell free translationassays. The inhibitory properties of oligomers containing a mixture ofuncharged and cationic linkages can be enhanced between, approximately,five to 100 fold in cell free translation assays.

III. Treatment of Muscle Wasting

The invention provides methods for treatment of muscle wasting with anantisense oligonucleotide directed against a nucleic acid sequenceencoding myostatin, and is based on the discovery that a stable,substantially uncharged phosphorus-linked morpholino antisense compound,characterized by high Tm, capable of active or facilitated transportinto cells, and capable of binding with high affinity to a complementaryor near-complementary myostatin nucleic acid sequence, can beadministered to a human subject or patient and inhibit expression ofmyostatin by muscle cells resulting in increased of muscle growth.

In vivo administration of a myostatin antisense oligomer to a subjectusing the methods described herein can result in an improved muscle massfor the patient, with the extent improvement dependent upon dose andfrequency of myostatin antisense oligomer administration and the generalcondition of the subject.

In preferred applications of the method the subject is a human patientdiagnosed as having degenerated or reduced muscle mass secondary to aprimary indication or disease state such as cancer, acquired immunedeficiency syndrome (AIDS) or muscular dystrophy. The patient may alsobe one who does not have a muscle wasting disease but be in need ofmaintaining or increasing muscle mass and tone to offset normal loss ofmuscle mass as one ages, or muscle loss due to an extended period ofinactivity.

As a first step in the treatment method, the patient is tested formyostatin levels, typically using a standard assay for measuring serummyostatin levels. See, for example, Yarasheski, et al., Schulte et al.,and Kirk, et al. for methods and reagents for measuring serummyostatin-immunoreactive levels. If the measured levels are above aselected threshold level for normal average individuals, and typicallymore than 10-20% above the selected threshold, or if the patientotherwise presents with obvious muscle wasting, the patient is acandidate for the treatment method. Normal threshold values may bedetermined for normal healthy individuals within a certain gender and/orage bracket, e.g., men in the 20-35 years old age group, but moretypically, values for normal patients in the same category as the testpatients are preferred, except for very elderly patients, e.g., aboveage 70. Alternatively, levels of myostatin transcript may be determinedusing the heteroduplex detection method described below.

In another embodiment, the treatment is applied to patients who arelikely candidates for loss of muscle, e.g., those who are subject toextended periods of inactivity, even though measured levels of myostatinmay be within a normal range.

Treatment Regimens

After identifying the patient as a treatment candidate, the patient isadministered an amount of the antisense compound effective to raisemeasured myostatin levels over a suitable response period, e.g., 1-3days following administration of the antisense compound.

In accordance with the invention, effective delivery of an oligomerantisense to a human subject may include, but is not limited to, varioussystemic routes, including oral and parenteral routes, e.g., intravenous(IV), subcutaneous, intraperitoneal (IP), and intramuscular; as well asinhalation and transdermal delivery. It is appreciated that any methodseffective to deliver a myostatin antisense oligomer to into thebloodstream of a subject are also contemplated. Transdermal delivery ofantisense oligomers may be accomplished by use of a pharmaceuticallyacceptable carrier adapted for e.g., topical administration. One exampleof morpholino oligomer delivery is described in PCT patent applicationWO 97/40854, incorporated herein by reference.

In one preferred embodiment, the oligomer is a phosphorodiamidatemorpholino oligomer (PMO), contained in a pharmaceutically acceptablecarrier, and delivered orally. In a further aspect of this embodiment, amorpholino myostatin antisense oligonucleotide is administered atregular intervals for a short time period, e.g., daily for two weeks orless. However, in some cases the antisense oligomer is administeredintermittently over a longer period of time.

Typically, one or more doses of antisense oligomer are administered,generally at regular intervals for a period of about one to two weeks.Preferred doses for oral administration are from about 1 mgoligomer/patient to about 100 mg oligomer/patient (based on an adultweight of 70 kg). In some cases, doses of greater than 100 mgoligomer/patient may be necessary. For IV administration, the preferreddoses are from about 0.5 mg oligomer/patient to about 10 mgoligomer/patient (based on an adult weight of 70 kg). The antisensecompound is generally administered in an amount sufficient to result ina peak blood concentration of at least 200-400 nM antisense oligomer.Greater or lesser amounts of oligonucleotide may be administered asrequired and maintenance doses may be lower.

At regular intervals during the treatment method, e.g., 1-3 daysfollowing administration of the antisense compound, the patient ismonitored for changes in myostatin levels, e.g., serummyostatin-immunoreactive protein. The dose of antisense compoundadministered to the patient should be such as to reduce measuredmyostatin level over the initially measured level. Typically a decreasein measured myostatin level of at least 10-20% over initial levels isdesired. If the initial measured levels were above a selected thresholdfor normal healthy individuals, the dose of antisense compound ispreferably adjusted to bring the myostatin level within this normalrange, and the treatment is continued, as needed to maintain myostatinlevels within this range. Diagnosis and monitoring of muscle wastinggenerally involves monitoring weight loss due to increased skeletalmuscle breakdown (Wallace and Schwartz 2002). Such methods may bequalitative or quantitative.

In some cases, the treatment regimen will include further interventionsuch as radiation therapy, immunotherapy and/or additional chemotherapy.Such treatment may occur prior to, during or subsequent toadministration of the chemotherapeutic agent and myostatin antisenseoligomer.

Materials and Methods Phosphorodiamidate Morpholino Oligomers

PMO were synthesized and purified at AVI BioPharma, Inc. (Corvallis,Oreg.) as previously described (Geller, Deere et al. 2003, Summerton andWeller, 1997), dissolved in water, filtered through a 0.2 μM membrane(HT Tuffryn™, Gelman Sciences, Inc., Ann Arbor, Mich.), and stored at 4°C. Exemplary sequences of PMO used are shown in Table 2. Theconcentration of PMO was determined spectrophotometrically by measuringthe absorbance at 260 nm and calculating the molarity using theappropriate extinction coefficient.

A schematic of a synthetic pathway that can be used to make morpholinosubunits containing a (1 piperazino) phosphinylideneoxy linkage is shownin FIG. 3; further experimental detail for a representative synthesis isprovided in Materials and Methods, below. As shown in the Figure,reaction of piperazine and trityl chloride gave trityl piperazine (1a),which was isolated as the succinate salt. Reaction with ethyltrifluoroacetate (1b) in the presence of a weak base (such asdiisopropylethylamine or DIEA) provided 1-trifluoroacetyl-4-tritylpiperazine (2), which was immediately reacted with HCl to provide thesalt (3) in good yield. Introduction of the dichlorophosphoryl moietywas performed with phosphorus oxychloride in toluene.

The acid chloride (4) is reacted with morpholino subunits (moN), whichmay be prepared as described in U.S. Pat. No. 5,185,444 or in Summertonand Weller, 1997 (cited above), to provide the activated subunits(5,6,7). Suitable protecting groups are used for the nucleoside bases,where necessary; for example, benzoyl for adenine and cytosine,isobutyryl for guanine, and pivaloylmethyl for inosine. The subunitscontaining the (1 piperazino) phosphinylideneoxy linkage can beincorporated into the existing PMO synthesis protocol, as described, forexample in Summerton and Weller (1997), without modification.

Although the invention has been described with reference to particularembodiments and applications, it will be appreciated that variouschanges and modifications may be made without departing from theinvention.

1. A method of treating skeletal muscle mass deficiency in a humansubject comprising (a) measuring blood or tissue levels of myostatin inthe subject, (b) administering to the subject, amyostatin-expression-inhibiting amount of a substantially unchargedantisense compound (i) composed of morpholino subunits andphosphorus-containing intersubunit linkages joining a morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.(ii) capable of uptake by target muscle cells in the subject, (iii)containing between 10-40 nucleotide bases, and (iv) having a basesequence effective to hybridize to an expression-sensitive region ofprocessed or preprocessed human myostatin RNA transcript, identified, inits processed form, by SEQ ID NO:6, (c) by said administering, formingwithin target muscle cells in the subject, a base-paired heteroduplexstructure composed of human myostatin RNA transcript and the antisensecompound and having a Tm of dissociation of at least 45° C., therebyinhibiting expression of myostatin in said cells, (d) at a selected timefollowing said administering, measuring a blood or tissue level ofmyostatin in the subject, and (e) repeating said administering, usingthe myostatin levels measured in (d) to adjust the dose or dosingschedule of the amount of antisense compound administered, if necessary,so as to reduce measured levels of myostatin over those initiallymeasured and to maintain such levels of myostatin measured in step (d)within a range determined for normal, healthy individuals.
 2. The methodof claim 1, wherein the myostatin value measured in step (a) is above aselected threshold for normal healthy people.
 3. The method of claim 1,wherein the morpholino subunits in the compound administered are joinedby phosphorodiamidate linkages, in accordance with the structure:

where Y₁═O, Z═O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkylamino, including dialkylamino.
 4. The method of claim 1, wherein thecompound is administered as a conjugate of the compound and anarginine-rich polypeptide effective to promote uptake of the compoundinto muscle target cells.
 5. The method of claim 2, in which at least 2and no more than half of the total number of intersubunit linkages arepositively charged at physiological pH.
 6. The method of claim 2,wherein said morpholino subunits are joined by phosphorodiamidatelinkages, in accordance with the structure:

where Y₁═O, Z═O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X for the uncharged linkages is alkyl, alkoxy,thioalkoxy, or an alkyl amino of the form wherein NR₂, where each R isindependently hydrogen or methyl, and for the positively chargedlinkages, X is 1-piperazine.
 7. The method of claim 4, wherein thearginine rich peptide has one of the sequences identified as SEQ IDNOS:7-9, and the arginine-rich peptide is covalently coupled at its Cterminus to the 5′ end of the antisense compound.
 8. The method of claim1, wherein said antisense compound is effective to hybridize to a targetregion at or adjacent the start site of the processed human myostatintranscript, the compound has a base sequence that is complementary to atarget region containing at least 12 contiguous bases in a processedhuman myostatin transcript identified by SEQ ID NO:10, and formation ofthe heteroduplex in step (c) is effective to block translation of saidprocessed transcript.
 9. The method of claim 8, wherein the antisensecompound includes a base sequence identified by the sequence SEQ IDNO:1.
 10. The method of claim 1, wherein the antisense compound iseffective to hybridize to a splice site of preprocessed human myostatintranscript, and has a base sequence that is complementary to at least 12contiguous bases of a splice site in a preprocessed human myostatintranscript, and formation of the heteroduplex in step (c) is effectiveto block processing of a preprocessed myostatin transcript to produce afull-length, processed myostatin transcript.
 11. The method of claim 10,wherein the splice site in the preprocessed myostatin transcript has asequence selected from the group consisting of: SEQ ID NOS: 11-14. 12.The method of claim 11, wherein the antisense compound includes a basesequence selected from the group consisting of: SEQ ID NOS: 2-5.
 13. Themethod of claim 1, wherein said administering and measuring is carriedout for a selected period of at least 2 weeks.
 14. The method of claim1, wherein said administering is by oral administration.
 15. Anantisense composition for use in increasing skeletal muscle mass in ahuman subject comprising a substantially uncharged antisense compound(i) composed of morpholino subunits and phosphorus-containingintersubunit linkages joining a morpholino nitrogen of one subunit to a5′ exocyclic carbon of an adjacent subunit. (ii) capable of uptake bytarget muscle cells in the subject, (iii) containing between 10-40nucleotide bases, and (iv) having a base sequence effective to hybridizeto an expression-sensitive region of processed or preprocessed humanmyostatin RNA transcript, identified, in its processed form, by SEQ IDNO:6.
 16. The composition of claim 15, wherein the morpholino subunitsin the compound are joined by phosphorodiamidate linkages, in accordancewith the structure:

where Y₁═O, Z═O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkylamino, including dialkylamino.
 17. The composition of claim 15, whichincludes said compound conjugated to an arginine-rich polypeptideeffective to promote uptake of the compound into target muscle cells.18. The composition of claim 15, in which a least 2 and no more thanhalf of the total number of intersubunit linkages are positively chargedat physiological pH.
 19. The composition of claim 18, wherein saidmorpholino subunits are joined by phosphorodiamidate linkages, inaccordance with the structure:

where Y₁═O, Z═O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X for the uncharged linkages is alkyl, alkoxy,thioalkoxy, or an alkyl amino of the form wherein NR₂, where each R isindependently hydrogen or methyl, and for the positively chargedlinkages, X is 1-piperazine.
 20. The composition of claim 17, whereinthe arginine rich peptide has one of the sequences identified as SEQ IDNOS: 7-9.
 21. The composition of claim 17, arginine-rich peptide iscovalently coupled at its C terminus to the 5′ end of the antisensecompound.
 22. The composition of claim 15, wherein said antisensecompound is effective to hybridize to a target region at or adjacent thestart site of the processed human myostatin transcript, the compound hasa base sequence that is complementary to a target region containing atleast 12 contiguous bases in a processed human myostatin transcriptidentified by SEQ ID NO:10, and formation of the heteroduplex in step(c) is effective to block translation of said processed transcript. 23.The composition of claim 22, wherein the antisense compound includes abase sequence identified by the sequence SEQ ID NO:
 1. 24. Thecomposition of claim 15, wherein the antisense compound is effective tohybridize to a splice site of preprocessed human myostatin transcript,and has a base sequence that is complementary to at least 12 contiguousbases of a splice site in a preprocessed human myostatin transcript, andformation of the heteroduplex in step (c) is effective to blockprocessing of a preprocessed myostatin transcript to produce afull-length, processed myostatin transcript.
 25. The composition ofclaim 24, wherein the splice site in the preprocessed myostatintranscript has a sequence selected from the group consisting of: SEQ IDNOS: 11-14.
 26. The composition of claim 25, wherein the antisensecompound includes a base sequence selected from the group consisting of:SEQ ID NOS: 2-5.
 27. A method of measuring myostatin expression levelsin a mammalian subject, comprising (a) administering to the subject, amyostatin-expression-inhibiting amount of a substantially unchargedantisense compound (i) composed of morpholino subunits andphosphorus-containing intersubunit linkages joining a morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.(ii) capable of uptake by target muscle cells in the subject, (iii)containing between 10-40 nucleotide bases, (iv) having a base sequenceeffective to hybridize to a region of processed or preprocessed humanmyostatin RNA transcript, identified, in its processed form, by SEQ IDNO:6, and (v) capable of forming with a such myostatin RNA transcript, abase-paired heteroduplex structure composed of human myostatin RNAtranscript and the antisense compound and having a Tm of dissociation ofat least 45° C., (b) within about 8-72 hours following saidadministering, analyzing a body-fluid sample obtained from the subjectfor the presence of a heteroduplex composed of said antisense compoundand a complementary region of said myostating RMA transcript, todetermine the concentration of transcript in said sample.